Hepatitis E Virus in Young Pigs in Finland and Characterization of the Isolated Partial Genomic Sequences of Genotype 3 HEV

Abstract

Hepatitis E virus (HEV) infections occur in swine worldwide. The porcine infection is usually subclinical, but HEV genotypes 3 and 4 are zoonotic agents that cause sporadic, indigenous human cases of hepatitis E. The aims of this study were to investigate the occurrence and dynamics of HEV infections in young pigs by analyzing a total of 273 fecal samples collected from six farrowing farms, to genetically characterize the HEV isolates obtained, and to examine the phylogenetic relationships of HEV isolates occurring at different swine farms in Finland. Fecal shedding of HEV of individual piglets was followed at two farms that were selected from five farms identified as HEV RNA positive. Excretion of HEV was detected in 87.5% of the piglets during the survey. Piglets contracted primary HEV infection 3–8 weeks after weaning, and at the time they were transferred to fattening farms, practically all (96.6%) of the pigs with a sample available at this occasion still excreted the virus. According to phylogenetic analysis, all HEV isolates obtained belonged to HEV genotype 3, subtype e, and a separate, farm-specific isolate originated from 10 of 11 farms examined. The results of our study show that HEV infections are highly common in young pigs, and HEV RNA–positive pigs enable HEV transmission from farrowing to fattening farms, creating a possible risk of infection for pig handlers, and that genetic variations in HEVs originating from different farms occur.

Introduction

Hepatitis E virus (HEV), the sole member of the genus Hepevirus in the family Hepeviridae (Emerson et al., 2004), is the causative agent of hepatitis E. Four HEV genotypes infecting mammals all belong to the same serotype (Lu et al., 2006). Genotypes 1 and 2 are the major causes of enterically transmitted hepatitis in the developing countries, where HEV also occurs as epidemics (Lu et al., 2006). In the industrialized countries, sporadic, indigenous human cases of hepatitis E are caused by genotypes 3 and 4, which are zoonotic agents and also common in swine populations throughout the world (Dalton et al., 2008). The porcine HEV infection is usually asymptomatic, and the infection route is believed to be fecal–oral, as in humans (Meng et al., 1998; Halbur et al., 2001; Bouwknegt et al., 2011). However, the sources of the infection in pigs, as well as the sources and routes of autochthonous human infections in developed countries, are still not clearly understood (Dalton et al., 2008; Andraud et al., 2013).

In humans, foodborne HEV infections of swine origin have been demonstrated to occur (Matsubayashi et al., 2008; Banks et al., 2010; Colson et al., 2010; Miyashita et al., 2012). HEV RNA has been detected in pig livers sold in grocery stores (Yazaki et al., 2003; Bouwknegt et al., 2007; Feagins et al., 2007; Wenzel et al., 2011), as well as in samples collected from slaughterhouses (Leblanc et al., 2010; Berto et al., 2012; Di Bartolo et al., 2012). Recently, concern has been raised by the considerable morbidity and mortality that genotype 3 HEV infection causes in immunocompromised individuals, including chronic infections in HEV-infected organ transplant recipients (Dalton et al., 2008).

In our previous study, we found acute HEV infections in pigs in the beginning of the fattening period (Kantala et al., 2013), and in the current study, we aimed at broadening the investigation of dynamics of HEV infections in the pig production chain. We determined what age piglets shed HEV in their feces by investigating HEV infections in pigs 2.5–12 weeks of age at 6 farrowing farms in Finland to enable us to estimate the risk of HEV infection they constitute for humans, especially persons working at swine farms. The aim was also to examine the phylogenetic relationships of HEV strains occurring at different swine farms.

Materials and Methods

Study design and sampling protocols

The study consisted of three parts: (1) identification of HEV RNA positive farms in 2009, (2) follow-up studies of HEV occurrence and dynamics in young pigs, and (3) phylogenetic analysis of HEV strains occurring at different swine farms (Fig. 1). In September–December 2009, 6 farrowing farms (farms 1–6) were studied for the presence of HEV RNA to identify HEV-positive farms. We collected fecal samples from 10 individual pigs per farm (total n=60), from 1 day to 15 weeks of age, and pooled fecal samples from 12 pens per farm (total n=72), housing pigs 1 week to 4 months of age. Pooled samples were collected from the floors of pens and individual samples directly from the rectums of pigs, using clean plastic gloves. Two farms (1 and 2) were selected for further follow-up studies that were conducted in October–December, 2010, to examine the age at which fecal shedding of HEV begins in piglets, and the length of time of the shedding. At both farms, 20 piglets, from 4 (farm 1) and 5 (farm 2) litters, were included in the follow-ups. Individual fecal samples (total n=149) were collected from the piglets four times, every 2–4 weeks, beginning at the age of 2–4 weeks (mean=26 days). All fecal samples were transported to the laboratory within 24 h in a cold box, and stored at −20°C until further processed.

FIG. 1.

Description of the farms that were included in the different parts of the study and of the years the fecal samples were collected from each of them. The “X” shows the farms from which samples were included in each part of the study, and the arrows show which samples from each of the farms were included in the phylogenetic analysis. The additional samples from 2005 and 2007 that were included in the phylogenetic analysis are shown on the right. Samples from pigs from farms 8–12 were collected from a swine test station in our previous study in 2007 (Kantala et al., 2013) and were selected for the phylogenetic analysis based on being hepatitis E virus (HEV) RNA–positive during the first week the pigs spent at the station, suggesting that the infections were contracted at the farms of origin of the pigs, and the sequences represent the HEV isolates that occurred at the farms.

To achieve a more comprehensive view of the genetic diversity of the HEV strains occurring at different farms, 10 samples with previously obtained ORF1 and/or ORF2 sequences from our previously published and unpublished studies were added to the phylogenetic analysis in addition to the samples from farms 1–6. From our earlier study (Kantala et al., 2013), previously published ORF1 sequences (GenBank accession numbers JN585124–JN585127; 3 identical sequences were reported under the same accession number) from 6 samples collected in 2007 from five farms (farms 8–12), and from our previously unpublished studies, 1 sample collected in 2007 from farm 8 and 3 samples collected in 2005 from farms 1 and 7 were included in the analysis (Fig. 1).

HEV RNA detection

RNA was extracted from all fecal samples from 140 μL of 10% suspensions in 0.1 M Tris-HCl saline buffer/phosphate-buffered saline buffer using QIAamp^® Viral RNA Mini Kit (Qiagen GmbH, Hilden, Germany) to reach a final volume of 60 μL. HEV RNA was detected by real-time one-step reverse transcription polymerase chain reaction (RT-PCR) targeting a 68-base pair (bp) fragment of the structural gene of HEV in ORF2, using the modified protocol with primers HEV2F and HEV2R, and probe JVHEVP (Table 1) described previously (Kantala et al., 2013).

Table 1.

Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Primers and Probe, and Conventional RT-PCR and Nested RT-PCR Primers Used for PCRs and Sequencing

Primer/probe	Target gene	PCR method	Sequence	Location	Polarity	Reference
HEV2F	Structural gene	Real-time	5′-GTGGTTTCTGGGGTGAC-3′	5262—5278	Positive	Kantala et al., 2009
HEV2R	(ORF2)	RT-PCR	5′-GGGGTTGGTTGGATGAATA-3′	5311—5329	Negative
JVHEVP			5′-TGATTCTCAGCCCTTCGC-3′	5284—5301		Jothikumar et al., 2006
ISP	RNA polymerase	Conventional	5′-GTATTTCGGCCTGGAGTAAGAC-3′	4232—4253	Positive	Zhai et al., 2006
lmR	(ORF1)	RT-PCR	5′-GTCCAGGCCGAYCTAACTAA-3′	4511—4530	Negative	Kantala et al., 2013
3156	Structural gene	Conventional	5′-AAYTATGCMCAGTACCGGGTTG-3′	5687—5708	Positive	Meng et al., 1997
3157	(ORF2)	Nested RT-PCR	5′-CCCTTATCCTGCTGAGCATTCTC-3′	6395—6417	Negative
3158			5′-GTYATGYTYTGCATACATGGCT-3′	5972—5993	Positive
3159			5′-AGCCGACGAAATYAATTCTGTC-3′	6298—6319	Negative

The sequence positions refer to the hepatitis E virus prototype Burma strain (M73218).

Conventional RT-PCR, gel electrophoresis, and nucleotide sequencing

All samples positive for HEV RNA by real-time RT-PCR were amplified with 2 conventional RT-PCR methods: 1-step RT-PCR targeting a 298-bp fragment of the RNA polymerase gene in ORF1 with primers ISP (Zhai et al., 2006) and lmR (Kantala et al., 2013), and nested RT-PCR targeting a 348-bp fragment of the structural gene in ORF2 with external primers 3156 and 3157 and internal primers 3158 and 3159 (Meng et al., 1997) (Table 1). The one-step RT-PCR with primers ISP and lmR was performed as described by Kantala et al. (2013), as was the first-round RT-PCR of the nested PCR with primers 3156 and 3157 with the exception of a lower annealing temperature of 52°C. The first-round RT-PCR product of the nested PCR was then amplified in second-round PCR, using 0.5 μM primers 3158 and 3159, HotStarTaq^® Plus Master Mix Kit (Qiagen) according to the manufacturer's instructions, and 1 μL of the first-round RT-PCR product. The reaction was carried out as follows: initial activation at 95°C for 5 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min.

The PCR products were separated on 2% MetaPhor® agarose gel (Lonza, Basel, Switzerland) stained with ethidium bromide. If this was not successful, a band-stab PCR technique (Bjourson and Cooper, 1992) was used. All PCR products were sequenced at the DNA Sequencing Service, Institute of Biotechnology, University of Helsinki using an Applied Biosystems ABI3130XL Genetic Analyzer or an ABI3730 DNA Analyzer.

Phylogenetic analysis

The sequences obtained from the samples collected from the six farms in 2009–2010, together with the additional sequences from our previously published and unpublished studies, were compared with HEV sequences in NCBI GenBank using the BLASTN program, and with each other using the BioEdit program (http://www.mbio.ncsu.edu). For phylogenetic trees, 16 additional, previously reported HEV sequences from elsewhere were used (Fig. 2). Multiple alignment analysis and bootstrap value calculation for the nucleotide (nt) sequences were conducted using ClustalX 2.1 program. The phylogenetic trees were constructed using NJplot program with a neighbor-joining method, using Kimura's correction for multiple substitutions.

FIG. 2.

Phylogenetic trees based on a fragment of 216 nucleotides (nt) within the RNA polymerase gene in ORF1 (A) and on a fragment of 268 nt within the structural gene in ORF2 (B), showing the sequences from this study together with 16 previously reported hepatitis E virus isolates from elsewhere representing genotypes 1–4 from GenBank. Bootstrap values of 30% and above are shown. The names of the isolates from which a previously unreported sequence was obtained in this study are in boldface. The sequences from this study are framed according to the farms they originated from, and the names of the farms from which both ORF1 and ORF2 sequences were obtained and included in the phylogenetic trees are in boldface.

Results

Occurrence and dynamics of HEV infections in young pigs

Originally, 5 of 6 (83.3%) farrowing farms that were investigated to determine HEV RNA–positive farms had at least 1 pooled fecal sample positive for HEV RNA (farms 1–5). The pigs in the positive pens were 3–14 weeks of age (Table 2). Only 1 of 60 individually sampled pigs, a 2-month-old pig from farm 3, was positive for HEV RNA. Two positive farms, 1 and 2, were selected for further individual sampling.

Table 2.

Results of the Hepatitis E Virus (HEV) RNA Detection from Pooled Fecal Samples Collected from Six Farrowing Farms in 2009

	No. of HEV RNA—Positive samples/no. of samples tested (%)
Age of pigs in the pen	Farm 1	Farm 2	Farm 3	Farm 4	Farm 5	Farm 6	Total per age group
0—2 wks	0/2	0/0	0/0	0/4	0/1	0/3	0/12
3—4 wks	0/4	0/3	0/2	1/2 (50.0)	0/3	0/1	1/15 (6.7)
5—6 wks	0/2	0/0	0/1	1/2 (50.0)	0/0	0/4	1/9 (11.1)
7—8 wks	1/1 (100.0)	0/1	2/6 (33.3)	0/0	0/4	0/0	3/12 (25.0)
>8 wks	2/3 (66.7)	3/4 (75.0)	1/3 (33.3)	2/3 (66.7)	2/4 (50.0)	0/4	10/21 (47.6)
Total per farm	3/12 (37.5)	3/8^a (37.5)	3/12 (25.0)	4/11^a (36.4)	2/12 (16.7)	0/12	15/67 (22.4)

Due to poor quality, some samples had to be excluded from the analysis, resulting in a total number of <12 samples tested in farms 2 and 4.

At each of the farms 1 and 2, all piglets were negative in the first 2 samplings on days 1 and 13 of the follow-up (Table 3). The first HEV RNA–positive piglets, at least one in each of the five litters (e–i), were detected at farm 2 in the third sampling. At farm 1, positive pigs were only found in the fourth sampling. In all, 28 of 29 (96.6%) pigs were positive at the end of the follow-up: all 20 pigs at farm 1 and 8 of 9 (88.9%) pigs at farm 2. At farm 2, the last sampling was delayed by 1 week for reasons beyond our control, and thus the last sample was not available from 11 pigs that had already been sent to a fattening farm. In three cases at farm 2, the same pig had two consecutive positive samples.

Table 3.

Results of Hepatitis E Virus (HEV) RNA Detection by Real-Time Reverse Transcription Polymerase Chain Reaction in Fecal Samples of Individual Pigs in the Follow-Ups at Farms 1 and 2 in 2010

		Farm 1						Farm 2
		HEV RNA detection (Ct values of the positive samples)						HEV RNA detection (Ct values of the positive samples)
		Day 1,	Day 13,	Day 34,	Day 55,			Day 1,	Day 13,	Day 28,	Day 55,
Pig ID	Litter ID	age 2–4 wks	age 4–6 wks	age 7–9 wks	age 10–12 wks	Pig ID	Litter ID	age 3–4 wks	age 5–6 wks	age 7–8 wks	age 11–12 wks
1	a	−	−	−	+ (31.93)	21	e	−	−	−	+ (33.65)
2	a	−	−	−	+ (22.06)	22	e	−	−	−	m
3	a	−	−	−	+ (34.94)	23	e	−	−	−	+ (33.32)
4	a	−	−	−	+ (27.50)	24	e	−	−	+ (36.07)	m
5	b	−	−	−	+ (31.73)	25	f	−	−	+ (34.01)	+ (35.09)
6	b	−	−	−	+ (32.40)	26	f	−	−	−	−
7	b	−	−	−	+ (34.00)	27	f	−	−	+ (33.06)	m
8	b	−	−	−	+ (36.00)	28	f	−	−	+ (33.78)	m
9	b	−	−	−	+ (32.70)	29	g	−	−	−	m
10	c	−	−	−	+ (33.70)	30	g	−	−	−	+ (34.20)
11	c	−	−	−	+ (32.30)	31	g	−	−	−	m
12	c	−	−	−	+ (35.40)	32	g	−	−	+ (37.51)	m
13	c	−	−	−	+ (33.10)	33	h	−	−	+ (35.37)	+ (23.82)
14	c	−	−	−	+ (33.40)	34	h	−	−	+ (37.66)	m
15	d	−	−	−	+ (31.30)	35	h	−	−	+ (26.55)	+ (20.88)
16	d	−	−	−	+ (36.10)	36	h	−	−	+ (34.35)	m
17	d	−	−	−	+ (32.80)	37	i	−	−	−	m
18	d	−	−	−	+ (33.90)	38	i	−	−	−	+ (35.03)
19	d	−	−	−	+ (34.00)	39	i	−	−	+ (38.32)	m
20	d	−	−	−	+ (34.40)	40	i	−	−	−	+ (22.20)
Total no. of positive samples/samples taken		0	0	0	20/20 (100%)	Total no. of positive samples/samples taken		0	0	10/20 (50%)	8/9 (89%)

Ct, threshold cycle; +, positive; −, negative; m, missing sample.

Phylogenetic analysis of the genomic sequences

In addition to determining the phylogenetic relationships of the newly obtained isolates from this study among reported HEVs, we also investigated the diversity of isolates originating from six additional farms (Fig. 1). All sequenced isolates belonged to HEV genotype 3, subtype e according to Lu et al. (2006). From 45 HEV RNA–positive samples from farms 1–5 from 2009 and 2010, a total of 43 sequences were obtained. An ORF1 sequence was obtained from 27 (44.3%) and an ORF2 sequence from 16 (26.2%) samples: Both ORF1 and ORF2 sequences were obtained from 13 samples, only ORF1 sequences from 14 samples, and only ORF2 sequences from 3 samples. Four incomplete sequences were excluded from the analysis. Ten of the ORF1 sequences (KJ825678–KJ825682 and KJ825684–KJ825688) and eight of the ORF2 sequences (KJ825691–KJ825693 and KJ825695–KJ825699) differed from each other. From the sample that was collected in 2005 from farm 1, both ORF1 (KJ8256839) and ORF2 (KJ825694) sequences were obtained. From the isolates from the 6 additional farms (farms 7–12) (Fig. 1), 5 previously unreported, unique sequences (ORF1: KJ825689 and KJ825690, ORF2: KJ825700–KJ825702), and 1 sequence that was identical with 1 of our previously published sequences (JN585121) were obtained. In the phylogenetic trees, a total of 28 previously unpublished and 6 previously published ORF1 sequences, and 18 previously unpublished ORF2 sequences were included (Fig. 2).

Sequences within each of the farms 1–5 and 7–11 were arranged as separate clusters of their own in both phylogenetic trees, including sequences from the sample that was collected in 2005 from farm 1 that clustered with sequences from later samples from the same farm. The ORF1 sequence from the isolate from farm 12 clustered with sequences from farm 10. Several ORF1 and ORF2 sequences were obtained from 5 farms (1, 2, 7, 8, and 10), enabling sequence analysis within the clusters. At the nucleotide level, the maximum differences of the ORF1 and ORF2 sequences within these farms were 2.31% (5 of 216 nt) and 0.75% (2/268 nt), respectively. The ORF1 sequences from two consecutive samplings from one pig from farm 2 were identical. Between the isolates from all 11 farms, the differences of the ORF1 and ORF2 sequences were 6.02–12.57% and 3.36–11.57%, respectively.

On the amino acid level, all the ORF2 sequences were 100% identical, whereas between the ORF1 sequences, variation of 1–3 amino acids at positions 1440 (serine/S or proline/P), 1461 (glycine/G or valine/V), and 1462 (threonine/T or serine/S) in reference to the HEV prototype Burma strain (M73218) was observed. Sequences from the samples from 2009 to 2010 from farm 1 were grouped together, with amino acids S–G–T in the designated positions. The sequence from the sample that was collected in 2005 from farm 1 differed by one amino acid, with amino acids P–G–T, from the sequences from the later samples from the same farm. The sequences from all the other farms had amino acids P–G–S, except those from farm 8, which were grouped together farm-specifically with amino acids P–V–S at positions 1440, 1461, and 1462, respectively.

Discussion

We report here the first study of HEV occurrence in piglets at farrowing farms in Finland, as well as the results of our studies on genetic divergence of HEV strains occurring at different swine farms. The piglets began fecal shedding of HEV at 7–12 weeks of age, 3–8 weeks after weaning. Practically all pigs were shedding HEV at the time they were transferred to fattening farms, enabling HEV transmission from farrowing to fattening farms. The results confirmed our previous observation of HEV infections in pigs in the beginning of the fattening period, and, as the isolates from older pigs in that study (Kantala et al., 2013), all HEV isolates detected in the current study also belonged to HEV genotype 3, subtype e. All isolates that originated from the same farm belonged to the same cluster in phylogenetic analysis. From 10 of 11 farrowing farms, a separate cluster or isolate was obtained, and, interestingly, the isolates from a particular farm from 2005 and 2009–2010 belonged to the same cluster, suggesting that genetic variations in HEVs originating from different locations occurred.

At the farrowing farms, the piglets were weaned at ages of 3–4 weeks and moved to nursery units where they were raised until transferring to fattening farms at ages of 2–3 months. The first piglets must have contracted HEV infection shortly after weaning, since they began shedding HEV in their feces at the age of 7–8 weeks, and fecal HEV RNA excretion usually starts 1–2 weeks after infection (Meng et al., 1998; Halbur et al., 2001; Kasorndorkbua et al., 2004). However, 62.5% of the piglets only began shedding HEV at ages of 10–12 weeks. The varying age of onset of the infection or shedding may have resulted from the quantity and/or quality of colostrum providing maternal antibodies to the piglets. The duration of a piglet's passive immunity to HEV is related to the amount of sow antibodies (Meng et al., 1997; de Deus et al., 2008; Kanai et al., 2010). The maternal antibodies protect piglets from HEV infection approximately 30 days after birth in the majority of piglets, and in some, especially in piglets born to strongly seropositive sows, up to 60 days or even longer (Meng et al., 1997; Kasorndorkbua et al., 2003; de Deus et al., 2008; Kanai et al., 2010; dos Santos et al., 2009; Feng et al., 2011). In our study, however, the actual effect of maternal antibodies or possible HEV shedding of the sows on HEV infections of the piglets or their liability to infection could not be assessed, since HEV antibodies, viremia, and fecal shedding of HEV of the sows were not examined.

In the present survey, we found three pigs with two consecutive HEV RNA–positive samples. For two of them, the threshold cycle (Ct) value of the first positive sample was higher than the Ct value of the second sample, suggesting that the infection began with a lower level of HEV shedding, which was then increased by the time of the last sampling. This kind of pattern in the start of the infection was reported in an earlier study by Kanai et al. (2010), in which fecal shedding of HEV in pigs began with a low RNA copy number at the age of 30 days and the amount of RNA increased 10 days later and then either remained high or decreased gradually until ages of 100–110 days. For one pig in our study, the Ct value of the first positive sample was slightly lower than that of the last sample, demonstrating that the amount of HEV shed in the feces was already decreasing during the last sampling. Our study did not continue after the pigs were moved to the fattening farms, leaving the actual peak and the end of the viral shedding unconfirmed. However, at the end of the follow-ups, at the time of transfer to the fattening farms, the level of HEV shedding of virtually all pigs was high, making them a risk of infection not only to possible previously unexposed pigs at the fattening farm, but also to persons in contact with them on this occasion.

The highest percentage nucleotide differences of our ORF1 and ORF2 sequences within the same farm were 2.31% and 0.75%, respectively. Our ORF1-targeting primers targeted the 3′ end of ORF1, where the percentage nucleotide differences of HEV sequences at the isolate level are 7.0–12.1% according to Lu et al. (2006). Our ORF2-targeting primers targeted the middle region of ORF2, when Lu et al. (2006) showed that the nucleotide difference at the isolate level was 2.0–10.1% at the 5′ end and 5.6–14.8% at the 3′ end of ORF2. According to this, both the ORF1 and ORF2 sequences that originated from the same farm, even the isolate from 2005 from farm 1, all belonged to the same HEV isolate. Since the nucleotide differences between the various clusters in our study were 6.02–12.57% and 3.36–9.33% with the ORF1 and ORF2 primers, respectively, we suggest that a separate isolate originated from 10 of 11 farms in the study. This was also seen in the phylogenetic trees, where isolates from each of these farms formed a separate branch. Furthermore, a similar topology as clusters and branches in both trees was created by sequences that were obtained from the same sample. Less heterogeneity was observed at the amino acid level, since only two farms were separated according to the amino acid differences in the amplified ORF1 fragment. The homogeneity of the sequence from 2005 from farm 1 with sequences from 2009 to 2010 from the same farm indicate that the same HEV strain was maintained at the farm for 4 years, with one amino acid being replaced by another over time. To our knowledge, a follow-up this long at the same farm has not been reported before.

In our study, piglets were already infected by HEV at the farrowing farms, and commonly shedding the virus at the time they were transferred to the fattening farms, potentially transferring it there and creating a possible risk of infection for pig handlers. It is of future importance to determine the extent of this risk in addition to assessing what kind of control measures could be used for reducing HEV occurrence and transmission on farrowing farms to decrease this risk. Additionally, HEV-infected pigs that arrive at fattening farms may constitute a risk of infection for piglets arriving from HEV-unexposed farms. Infection at a later age during the fattening period constitutes a risk for entering of HEV into the food-chain in pork and pork-derived products at the time of slaughter. It is of future interest to investigate the occurrence of HEV infections in slaughter-aged pigs in Finland to determine the significance of this risk.

Footnotes

Acknowledgments

This study was supported by the Finnish Graduate School on Applied Bioscience (ABS), the project “Enteric Virus Emergence, New Tools” (EVENT, FP6-SP22-CT-2004-502571) funded by the European Union, the Mercedes Zachariassen Foundation, the Walter Ehrström Foundation, and the Finnish Foundation of Veterinary Research.

Disclosure Statement

No competing financial interests exist.

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